Homopolymers do not impact sequencing. The number of uniquely alignable reads is a function of the repeat content, so this will have an impact on productivity. With longer reads and paired-end sequencing, this may be less of an issue.

Can I use only 1 of the indexes of a dual-indexed library?

The new HiSeq v4 reagent kits now support dual indexing workflows without requiring the purchase of additional SBS agents. Sample prep for dual-indexed libraries requires that both indexes be present on the library. However, the second index does not need to be read during sequencing. A single-indexing workflow is supported on Illumina sequencing instruments, where only Index 1 is used. See the instrument user guide for more information about setting up an 8-base single-indexed sequencing run.

Reagents and Flow Cells

Illumina currently offers two SBS kits for the HiSeq: a 200-cycle kit and a 50-cycle kit. Both kits contain the same formulations and only differ in volume.

Are HiSeq/HiScanSQ flow cells compatible with the Cluster Station?

No. The HiSeq flow cell, also used on the HiScanSQ, must be clustered on a cBot equipped with an adapter plate suitable for the larger format of the flow cell.

Why is my SBS v3 reagent waste brown?

Due to an interaction with one of the v3 reagents, SRE, waste appears dark brown in color and has a stronger odor. This is normal. The change in waste color does not impact performance and is not toxic. You might see a discoloration on the funnel caps and SRE sipper line. Any spills will be dark brown in color as well.

How many additional cycles of SBS reagents do I need to calculate into the run for sequencing dual-indexed libraries?

For dual index paired-end runs, there are 23 additional cycles (index & chemistry only).For dual-index single-read runs, there are 16 additional cycles of indexing.For information about the number of SBS kits required on the HiSeq, HiScanSQ, or GAIIx, see the user guide for your instrument guide.

Can I split my 200-cycle kit for shorter runs?

Yes. You can split the 200-cycle SBS Kit into two equal volumes suitable for up to 101 cycles each. See the TruSeq SBS Kit Reagent Preparation Guide (200 Cycles) for instructions and storage requirements. If you need smaller volumes for shorter runs, Illumina recommends using the TruSeq SBS Kit (50 Cycles).

Do I need oil to load a flow cell on the HiSeq or HiScanSQ?

No. The HiSeq and HiScanSQ do not require immersion oil to properly load a flow cell in the way that the Genome Analyzer does. Instead, the flow cell is held in place by a vacuum, which removes air and replaces the need for immersion oil.

Has the tile layout or numbering changed due the wider lanes on Flow Cell v3?

No, the tile numbering is unchanged from the format introduced in HCS v1.3. However, when using Flow Cell v3, the tile numbering reflects the three-swath imaging pattern, where a 3 in the tile number represents the third swath.

Will I have enough reagents in my 200-Cycle SBS Kit to perform a 2x100 cycle run with indexing?

Can I use the same reagents on the HiSeq that I use on my Genome Analyzer?

No. Cluster Kits and SBS Kits for the HiSeq are not equivalent or compatible with the Genome Analyzer. Likewise, Cluster Kits and SBS Kits for the Genome Analyzer are not equivalent or compatible with the HiSeq.

Workflow

The TruSeq controls were not designed to be a qualitative metric of the efficiency of the various steps in the sample prep but rather an indicator of whether or not the step worked or not. Actual counts of the controls will vary based on sample type, input, etc.

In SAV (Sequencing Analysis Viewer), if you see counts for a particular control, this mean that this step has worked whereas if you see no counts (dark blue color), this step has likely failed. If the band has been cut across multiple insert sizes, you may see counts at different size increments.

What is the alternative SBS workflow?

TruSeq SBS v3 reagents enable an alternative workflow for loading all SBS reagents at the start of a 2x101-cycle sequencing run for both Read 1 and Read 2. Using this workflow might result in a slight increase in phasing in Read 2, which should not result in a decrease in quality.

How much additional time will be added to the current sequencing run time on HiSeq to support dual indexing?

When using v3 SBS reagents and the v3 flow cell on HiSeq, each additional index cycle is approximately 53 minutes per cycle (with 16 total indexing cycles) plus ~2 hours for the seven chemistry-only cycles for the PE workflow, resulting in ~16 hours of additional time for dual indexing on HiSeq.

What are the workflow changes for analysis/demultiplexing on the MiSeq, HiSeq, and GA for dual-indexed libraries compared to single-indexed libraries?

There are no changes for MiSeq analysis. HiSeq and GA data require an upgrade to CASAVA 1.8.2 to demultiplex dual-indexed libraries. It is also recommended to upgrade to SAV 1.8.4 or higher to use the new Index tab for real time demultiplexing information.

What is the optimal cluster density on the HiSeq?

The recommended maximum cluster density is 750,000-850,000 clusters/mm² when using Illumina's v3 cluster generation and sequencing reagents in combination with HCS v1.4.

Is a sample sheet/library sheet optional or mandatory for sequencing runs and analysis?

For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. When analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet when setting up the run in MCS.

Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) prior to performing library prep in order to confirm appropriate index combinations.

How many samples can be run on one flow cell?

Flow cells are designed for single-use. All eight lanes must be used at the same time. They can be used for the same sample or for dif­ferent samples. You can run eight samples at a time without multiplexing. With multiplexing, you can increase throughput to up to 12 samples per lane or up to 96 samples per flow cell.

For dual-indexed libraries, how many cycles are performed for index reads?

Dual-indexed runs on the HiSeq comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations. For more information, see the user guide for your sequencing instrument.

Do I need to use a cBot to cluster a HiSeq/HiScanSQ flow cell?

Yes. The HiSeq flow cell, also used on the HiScanSQ, requires the use of a cBot for clustering on the flow cell prior to sequencing.

How many cycles should be used during the Index Read for single-indexed libraries?

Index reads for single-read libraries use 7 cycle reads. Illumina does not support 6 cycle index reads for single-indexed libraries.

What is the workflow for dual indexing?

See the appropriate HiSeq instrument user guide for details on the loading of reagents with different workflows and which primers you need to use for your library type.

Read 1: An indexed Read 1 follows the standard Read 1 protocol using reagents provided in the TruSeq SBS Kit. The Read 1 sequencing primer is annealed to the template strand during the cluster generation process on the cBot (HP6 or HP10).

Index Read 1 (i7): Following the completion of Read 1, the run proceeds to Index Read preparation. The Read 1 product is removed and the Index 1 (i7) sequencing primer (HP8 or HP12) is annealed to the same template strand. The run proceeds through 8 cycles of sequencing to read the Index 1 (i7).

Index Read 2 (i5): For paired-end flow cells, the Index 1 (i7) Read product is removed and the template anneals to the grafted P5 primer on the surface of the flow cell. The run proceeds through an additional 7 chemistry-only cycles (no images are taken) followed by 8 cycles of sequencing to read Index 2 (i5).
For single-read flow cells, the Index 1 (i7) Read product is removed and the Index 2 (i5) sequencing primer (HP9) is annealed to the same template strand. The run proceeds through 8 cycles of sequencing to read the Index 2 (i5).

A PhiX spike-in employs a small amount of PhiX control in the same lane as a sample. This allows real time quality metrics as the PhiX is analyzed during the run. This is not recommended for sequencing a genome with high similarity to the PhiX genome, and does not allow for normalization of data in that lane as per a control lane.

Is initial cycle indexing possible on the HiSeq?

No, the system does not support an initial cycle indexing method. To ensure the highest quality data, Illumina recommends and supports a separate indexing read for multiplexed samples.

Can I monitor my HiSeq run remotely?

Illumina provides the Sequencing Analysis Viewer (SAV) software that can be run on a Windows PC to remotely monitor your run. The software does not allow any control over the run and requires that the PC is connected to the analysis server over the network. Another application you can use to monitor your run is SeqMonitor, which allows you to monitor your run using your iPhone or iPad.

Can I customize a HiSeq recipe?

With HCS v1.3 and later, you can customize a recipe to contain any number of reads. Reads can be indexed or non-indexed. However, Illumina does not guarantee the performance of custom recipes. Contact your Illumina Technical Support if you need assistance creating custom recipes.

Is a sample sheet/library sheet optional or mandatory for sequencing
runs and analysis?

For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a
sample sheet at the start of the run is optional. However, using a
sample sheet allows you to view data shown on the indexing tab in the
Sequencing Analysis Viewer (SAV) during the run. If you do not load a
sample sheet at the start of a run in HCS, you will not be able to
view indexing data in SAV. When analyzing indexed samples using CASAVA
v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet
when setting up the run in MCS.

Illumina recommends that you create the sample sheet using the
Illumina Experiment Manager (IEM) prior to performing library prep in
order to confirm appropriate index combinations.

Software

The instrument computer is a computational engine performing real time analysis of data. To avoid loss of data and other adverse effects, Illumina does not recommend installing any additional software with the exception of anti-virus software.

I got a warning message in HCS about the ARM9BoardSerialPort. What does this mean and what should I do?

The warning message "ARM9BoardSerialPort (ARM9CHEM): timed out waiting" indicates that an ARM9 communication time out has occurred. The ARM9 board is one of many components that communicate between the HiSeq and instrument computer. Messages related to an ARM9 time out are not necessarily indicative of a hardware issue, and do not impact the run or data quality.

If this message appears repeatedly, perform a normal stop on the current run, shut down the HCS/RTA software, and then power cycle the HiSeq and instrument computer to reestablish communication between the systems. Launch HCS and resume your run. Continue to monitor your run to make sure that the issue is resolved. If it appears that the run data is affected, contact Illumina Technical Support for further assistance.

How do I select the right number of index cycles and chemistry for sequencing dual-indexed libraries in HCS 1.5?

In order to perform dual-index sequencing in HCS 1.5, select the TruSeq Dual Index Sequencing Primer Box from the Index chemistry drop down menu on the recipe screen. This selection enables the use of the required chemistry for sequencing dual-indexed libraries, and must be used for sequencing any dual-indexed libraries (Nextera or TruSeq HT) regardless of which sequencing primers you will use for your run. Selecting any other setting will result in less than an eight-cycle index read.

Can I expect to see any changes in intensity with HCS v1.4 or HCS v1.5?

Intensity for the G channel is expected to be lower, but the rate of decay is much slower due to one of the new reagents in the TruSeq SBS Kit v3 - HS called SRE. Therefore, your data quality will not be impacted.

Can I save images from a run on the HiSeq or HiScanSQ?

No. Images are deleted automatically after they have been processed.

How is the instrument health upload option communicated to customers?

The first time the HCS 2.2 is launched, you will see a notification regarding instrument health data. This notification appears only once during the first initialization of the HCS, and will not appear again. Note that in pre-release, early access versions of HCS 2.0, this notification does not appear. However, instrument health agreement and notification is always available from Menu | Options | Tools, where you can also get more information and turn the option on or off.

How are images taken on the HiSeq or HiScanSQ?

Images are taken using a time delayed integration (TDI) line scanning optical system with four CCD sensors. The TDI line scanning system greatly increases throughput by maximizing camera utilization.

Can I save intensities from a run on the HiSeq or HiScanSQ?

Yes. The *.cif files can be saved and transferred to the analysis server over the network.

What is instrument health data, and where can I find more information about the option to send instrument health data to Illumina?

HCS v2.2 allows HiSeq instruments connected to the internet to send instrument health information to Illumina. This information is anonymous and includes only generic run metrics. This information is used by Illumina to help improve Illumina products. If you want to turn off this option or would like further information, see the Options menu in the HiSeq Control Software. You can find the Options menu under Menu, then Tools.

I got a warning message in HCS about the Tdi Scan. What does this mean and what should I do?

TDI Scan warning messages indicate an issue with image acquisition and storage; however, the system will automatically retry image capture to self-correct. TdiScan messages usually have no effect on the run other than slightly extended cycle times, and do not affect the run data as images are re-captured before continuing.

In the rare event that the retry threshold is exceeded, one imaging swath is skipped for one cycle. If this message occurs frequently, contact Illumina Technical Support for assistance.

What operating system is used on the instrument computer?

The HiSeq instrument computer employs 64-bit Windows Vista.

When I started my run, I got the HCS error message, “Must have at least one valid ETF to normalize/correct the failed ones.” What does this mean and what should I do?

This error message indicates a lack of fluorescence on the flow cell. To find focus at the start of a run, the software uses ETF, which is a focusing method that reads fluorescence from clusters on the flow cell. ETF must find fluorescence in at least one lane of the flow cell before the run can begin.

To correct this problem, perform a primer rehybridization. Re-annealing the Read 1 sequencing primer usually increases the fluorescence if clusters are present on the flow cell. Additionally, check the cBot plate to make sure that all reagents were delivered correctly and that the sequencing primer was appropriate for your library types. When you reload the flow cell on the HiSeq, confirm that the fluidics system is functioning correctly. If a primer rehybridization does not resolve the issue, contact Illumina Technical Support for further assistance.

Analysis

A quality score (or Q-score) is a prediction of the probability of an incorrect base call. Based on the Phred scale, the Q-score serves as a compact way to communicate very small error probabilities. Given a base call, X, the probability that X is not true, P(~X), is expressed by a quality score, Q(X), according to the relationship:Q(X) = -10 log10(P(~X))where P(~X) is the estimated probability of the base call being wrong.

A quality score of 10 indicates an error probability of 0.1, a quality score of 20 indicates an error probability of 0.01, a quality score of 30 indicates an error probability of 0.001, and so on.

During analysis, base call quality scores are written to FASTQ files in an encoded compact form, which uses only one byte per quality value. This method represents the quality score with an ASCII code equal to the value + 33.

If I use BaseSpace for run monitoring only, what files are sent to BaseSpace?

The files that are sent to BaseSpace are the InterOp folder, RunInfo.xml file, and RunParameters.xml file.

Yes. To convert zipped .bcl files, use bcl2fastq v1.8.4. This version can also convert non-zipped .bcl files.

How do I know if I need to throttle BaseSpace, and how do I apply throttling?

The BaseSpace Broker is designed to upload data to BaseSpace as soon as the data are generated on the HiSeq local drive. It will use as much bandwidth as is necessary to keep up with the data being produced. Under typical HiSeq run conditions, the upload of run data for storage and analysis will average less than 10Mbit/sec.

In most cases, throttling of the BaseSpace Broker data upload is not necessary. Throttling can be necessary if greater control over network bandwidth usage is required, such as sites where instruments share the network with other users or sites with limited upload speed. Throttling might be necessary in scenarios where the local network connectivity is temporarily lost and then restored. This interruption causes the BaseSpace Broker to suddenly consume more network bandwidth as it attempts to catch up with transfer of accumulated data. If no throttling is applied in such cases, the BaseSpace Broker might consume all available bandwidth on the network until the backlog of data are cleared. If throttling is applied and if the local network allows, Illumina recommends throttling to higher than the 10 Mbit/sec minimum specification. A recommended value of 20 Mbit/sec (approximately 3Mbytes/sec = 24Mbits/sec) allows the BaseSpace Broker enough bandwidth to recover, even if some delays in data transfer occur.

If throttling is needed, provide the following instructions to your local IT administrator:

Throttling of BaseSpace is performed on the HiSeq computer by application, rather than by IP address, as follows:

In Windows, open a cmd window and open the Local Policy Editor. Run the program gpedit.msc.

Select Only applications with this executable name and enter Illumina.BaseSpace.Broker.exe.

This policy applies to any source IP and target IP addresses. Click Next.

This policy applies to all ports and protocols. Click Finish.

How do I send run data to BaseSpace?

Run data can only be uploaded to BaseSpace if the BaseSpace option is selected during run setup in the HiSeq Control Software. See the HiSeq 2500 System User Guide (part # 15035786) for information on setting up a run with a connection to BaseSpace.

No. Thumbnail images are for visual inspection only to help diagnose problems with a run. They are not suitable for reanalysis.

Can I use MiSeq Reporter software to analyze TruSight DNA Amplicon data from a NextSeq or HiSeq system?

No, file directory structures are incompatible with MiSeq Reporter software. However, the TruSeq Amplicon App is available in BaseSpace and can be used to analyze the TruSeq Amplicon Cancer Panel, the TruSight Myeloid Sequencing Panel, and the TruSeq Custom Amplicon panels.

Does BaseSpace require a sample sheet?

If you choose to use BaseSpace only for run monitoring and your samples are not indexed, a sample sheet is not required. If you want to use BaseSpace for data storage and analysis, a sample sheet is required. The sample sheet can be in either HiSeq Analysis Software format or CASAVA format. When using BaseSpace, combining indexed and non-indexed samples on a flow cell is not possible.

What is bcl2fastq?

The bcl2fastq v1.8.4 conversion software is a separate piece of standalone software that is run on a Linux scientific computing system. The installer can be downloaded from the Illumina website. System requirements are outlined in the bcl2fastq User Guide (part # 15038058). If BCL files are zipped, then the use of the bcl2fastq v1.8.4 is required.

How do I use BaseSpace for run monitoring?

The Run Monitoring BaseSpace option allows you to remotely monitor a run in progress by logging in to your BaseSpace account. You need to select the Run Monitoring option during run setup. Then, log in to your BaseSpace account from anywhere and view your run in the BaseSpace version of Sequence Analysis Viewer (SAV).

How do I make sure that my HiSeq is ready to send data to BaseSpace?

To upload data to BaseSpace from a HiSeq, a minimum upstream connection of 10 Mbit/second per instrument is needed. Network speed can be assessed by using free online tools such as www.speedtest.net.

What data compression options are available for high output runs using HCS v2.2?

Two data compression options are zipping of BCL files and binning of Q-scores. Other run folder files are unchanged. These options are available during run setup in HCS v2.2. If you are using BaseSpace for data storage and analysis, BCL files are zipped automatically. Due to the size of the run folder with the extra cycles and shorter run durations, zipped BCL files are required for HiSeq v4 runs. This setting cannot be turned off. You can select or deselect Q-score binning depending on your preference.

Because run output has zipped BCL files, you must use the bcl2fastq v1.8.4 conversion software to perform BCL to FASTQ conversion on your local Linux analysis system. This tool is run on Linux and has the same syntax, options, and functions (including demultiplexing) as the configureBclToFastq.pl script of CASAVA. The only difference is that it can be used to analyze either zipped or non-zipped BCL files.

If you send your data to BaseSpace, BCL to FASTQ conversion and demultiplexing are performed automatically following the completion of the data upload.

What is deconvolution?

It is the ability to distinguish between two or more clusters that are in close proximity to each other.

How do I use BaseSpace for run monitoring only, such as SAV functionality in BaseSpace?

Run monitoring with BaseSpace is selected during run setup.

Is the output of bcl2fastq compatible with CASAVA v1.8.2?

If you are using CASAVA, it is compatible. However, bcl2fastq v1.8.4 must be used in place of the configureBcl2fastq step in CASAVA. The output of bcl2fastq v1.8.4 is in the fastq.gz file format organized into project and sample directories as specified in the sample sheet. This output is compatible with the configureAlignment and configureBuild components of CASAVA v1.8.2. The sample sheet format required for bcl2fastq v1.8.4 is equivalent to CASAVA v1.8.2 sample sheet format, and is described in the bcl2fastq v1.8.4 User Guide (part # 15038058).

If you are not using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.

Can I save .cif files in HiSeq v4 mode?

In HiSeq v4 mode .cif files cannot be saved. The option to save .cif files is available in other modes.

To remove the least reliable data from the analysis results, often derived from overlapping clusters, raw data are filtered to remove any reads that do not meet the overall quality as measured by the Illumina chastity filter. The chastity of a base call is calculated as the ratio of the brightest intensity divided by the sum of the brightest and second brightest intensities.

Clusters passing filter are represented by PF in analysis reports. Clusters pass filter if no more than one base call in the first 25 cycles has a chastity of < 0.6.

Do local proxies affect BaseSpace?

No testing has been performed on the effects of local proxies on BaseSpace access.

Can I use MiSeq Reporter software to analyze data from a HiSeq system?

No. File directory structures from a HiSeq system are incompatible with MiSeq Reporter software.

However, the TruSeq Amplicon App is available in BaseSpace and can be used to analyze the this kit.

What software do I use to analyze .cif files generated with HCS 2.x?

Where .cif files can be generated, you can use OLB 1.9.4.

Can I analyze .cif files with BaseSpace?

No, .cif files cannot be analyzed with BaseSpace. Additionally, it is not possible to output .cif files with HCS v2.2 on HiSeq v4 mode or Rapid Run mode with HiSeq v2 chemistry. The option to output .cif files is available in TruSeq v3 mode and Rapid Run mode with TruSeq chemistry.

How many swaths and tiles are on a 2-lane rapid run flow cell?

Scanning and analysis of a 2-lane rapid run flow cell creates 2 swaths per surface on 2 surfaces per lane. Each swath is divided into 16 tiles. For a 2-lane flow cell, there are a total of 128 tiles per flow cell.

What ports, domains, and encryption does BaseSpace use?

Contact your local IT administrator if local security policies have to be modified to allow access to BaseSpace. BaseSpace uses SSL/https port 443 and the domains *.basespace.illumina.com and *.s3.amazonaws.com. Data streaming to BaseSpace is encrypted using the AES256 standard and uses SSL for protection. More information on encryption can be found at http://blog.basespace.illumina.com/2011/12/13/basespace-security/

How do I merge data from 2 flow cells?

Using CASAVA: To merge data from different flow cells (different runs), use the configureBuild script in CASAVA v1.8.2. First, align the data (samples) from each flow cell separately using configureAlignment. Then, include each sample directory as an input directory in the configureBuild.pl command line. Input directories are specified by the -id option, as detailed on page 100 of the CASAVA v1.8.2 User Guide (Rev C).

If you are using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.

Using BaseSpace: BaseSpace includes a Sample Merge function that allows you to merge data from a single sample originating from different flow cells. This merging is performed before alignment analysis of the sample data.